Folate biosynthesis is an essential bacterial pathway that is absent in higher animals, and it has been a target of antibacterial agents for over 70 years. Sulfa drugs inhibit the enzyme dihydropteroate synthase (DHPS) in the pathway by acting as non-productive substrate analogs of p-aminobenzoic acid (pABA). However, the flexible pABA binding site is structurally susceptible to resistance mutations, and the sulfa drugs are rapidly becoming therapeutically ineffective. In contrast, the binding site of the second DHPS substrate, pterin-pyrophosphate, is buried in a conserved pocket that is less likely to tolerate mutations. We propose to generate new classes of potent DHPS inhibitors that specifically engage this pocket. These inhibitors can potentially be developed into novel therapeutic agents that still target folate synthesis but which avoid the problems of resistance. To generate effective inhibitors of any enzyme, it is crucial to understand the structure and mechanism of its active site. This information is largely absent for DHPS, and understanding how DHPS performs catalysis at the molecular level will be a central goal of the proposal. Another goal will be to understand the molecular and functional basis of sulfa drug resistance to avoid this problem in our future drug designs. This comprehensive approach will require the joint expertise of the two principal investigators in structural biology, medicinal chemistry, biochemistry, computational biology and microbiology. The project will also incorporate the relatively new technique of fragment based drug discovery to identify novel small inhibitory chemical scaffolds that can readily be elaborated by chemical synthesis. Exciting new data described in the application provide strong support that the project can realize its ultimate goal which is to rapidly develop new antimicrobials that minimize the emergence of drug resistance. Although we aim to develop broad-spectrum anti-infective agents, our microbiological screening will focus on S. aureus and P. jirovecii that have emerged as particularly problematical infectious disease agents in the U.S. in recent years.